Identification of anti-citrullinated osteopontin antibodies and increased inflammatory response by enhancement of osteopontin binding to fibroblast-like synoviocytes in rheumatoid arthritis.

BACKGROUND: Anti-citrullinated protein/peptide antibodies (ACPAs) are present in patients at onset and have important pathogenic roles during the course of rheumatoid arthritis (RA). The characteristics of several molecules recognized by ACPA have been studied in RA, but the positivity rate of autoantibodies against each antigen is not high, and the pathogenic mechanism of each antibody is not fully understood. We investigated the role of anti-citrullinated osteopontin (anti-cit-OPN) antibodies in RA pathogenesis. METHODS: Enzyme-linked immunosorbent assays on RA patients' sera were used to detect autoantibodies against OPN. Fibroblast-like synoviocytes (FLS) isolated from RA patients were used to test the binding activity and inflammatory response of OPN mediated by anti-cit-OPN antibodies, and their effect was tested using an inflammatory arthritis mouse model immunized with cit-OPN. Anti-cit-OPN antibody positivity and clinical characteristics were investigated in the patients as well. RESULTS: Using sera from 224 RA patients, anti-cit-OPN antibodies were positive in approximately 44% of RA patients, while approximately 78% of patients were positive for the cyclic citrullinated peptide (CCP2) assay. IgG from patients with anti-cit-OPN antibody increased the binding activity of OPN to FLSs, which further increased matrix metalloproteinase and interleukin-6 production in TNF-stimulated FLSs. Mice immunized with cit-OPN antibodies experienced severe arthritis. Anti-cit-OPN antibodies in RA patients decreased the drug survival rate of tumor necrosis factor (TNF) inhibitors, while it did not decrease that of CTLA4-Ig. CONCLUSIONS: Anti-cit-OPN antibodies were detected in patients with RA. IgG from patients with anti-cit-OPN antibodies aggravated RA, and anti-cit-OPN antibody was a marker of reduced the survival rate of TNF inhibitors in RA patients.

Role of Lysine-Specific Demethylase 1 in Metabolically Integrating Osteoclast Differentiation and Inflammatory Bone Resorption Through Hypoxia-Inducible Factor 1α and E2F1.

OBJECTIVE: Hypoxia occurs in tumors, infections, and sites of inflammation, such as in the affected joints of patients with rheumatoid arthritis (RA). It alleviates inflammatory responses and increases bone resorption in inflammatory arthritis by enhancing osteoclastogenesis. The mechanism by which the hypoxia response is linked to osteoclastogenesis and inflammatory bone resorption is unclear. This study was undertaken to evaluate whether the protein lysine-specific demethylase 1 (LSD1) metabolically integrates inflammatory osteoclastogenesis and bone resorption in a state of inflammatory arthritis. METHODS: LSD1-specific inhibitors and gene silencing with small interfering RNAs were used to inhibit the expression of LSD1 in human osteoclast precursor cells derived from CD14-positive monocytes, with subsequent assessment by RNA-sequencing analysis. In experimental mouse models of arthritis, inflammatory osteolysis, or osteoporosis, features of accelerated bone loss and inflammatory osteolysis were analyzed. Furthermore, in blood samples from patients with RA, cis-acting expression quantitative trait loci (cis-eQTL) were analyzed for association with the expression of hypoxia-inducible factor 1α (HIF-1α), and associations between HIF-1α allelic variants and extent of bone erosion were evaluated. RESULTS: In human osteoclast precursor cells, RANKL induced the expression of LSD1 in a mechanistic target of rapamycin-dependent manner. Expression of LSD1 was higher in synovium from RA patients than in synovium from osteoarthritis patients. Inhibition of LSD1 in human osteoclast precursors suppressed osteoclast differentiation. Results of transcriptome analysis identified several LSD1-mediated hypoxia and cell-cycle pathways as key genetic pathways involved in human osteoclastogenesis. Furthermore, HIF-1α protein, which is rapidly degraded by the proteasome in a normoxic environment, was found to be expressed in RANKL-stimulated osteoclast precursor cells. Induction of LSD1 by RANKL stabilized the expression of HIF-1α protein, thereby promoting glycolysis, in conjunction with up-regulation of the transcription factor E2F1. Analyses of cis-eQTL revealed that higher HIF-1α expression was associated with increased bone erosion in patients with RA. Inhibition of LSD1 decreased pathologic bone resorption in mice, both in models of accelerated osteoporosis and models of arthritis and inflammatory osteolysis. CONCLUSION: LSD1 metabolically regulates osteoclastogenesis in an energy-demanding inflammatory environment. These findings provide potential new therapeutic strategies targeting osteoclasts in the management of inflammatory arthritis, including in patients with RA.